ASSESSMENT OF SELECTED HEAVY METALS IN Perna viridis...

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ASSESSMENT OF SELECTED HEAVY METALS IN Perna viridis MUSSEL

AND SURFACE SEAWATER IN THE COASTAL AREA OF PASIR GUDANG,

MALAYSIA

NOR KAMILAH BINTI MUKTAR

A dissertation submitted in fulfilment of the

requirements for the award of the degree of

Master of Science (Chemistry)

Faculty of Science

Universiti Teknologi Malaysia

SEPTEMBER 2016

ii

DEDICATION

This is especially dedicated to my beloved parents (Mak and Abah), siblings, close

family members, dearest one and friends…

iii

ACKNOWLEDGEMENTS

First and foremost, I am grateful to Allah S.W.T. for giving me strength and

patience until I have completed this study and thesis writing successfully.

I would like to present a great appreciation and special thanks to my project

supervisor, Dr. Naji Arafat Mahat for his guidance, advice, encouragement and

support from the preliminary to the finishing level of my research project. My most

sincere thanks also go to him for the time spent and supervision.

Besides, I would like to express my gratitude to En. Azani for his help with

the analysis using FAAS. A lot of thanks to Pn. Zubaidha and Cik Aini for their help

and guidance using FIMS instrument as well as thanks to laboratory assistants, Pn.

Mariam and Pn. Hazelinda for all their kind help in the lab.

Finally, my sincere appreciation extends to my beloved parents and my

family for their encouragements, supports and everything. Also big thanks to my

friends for being helpful, supportive and cooperative, and to those who have helped

me directly or indirectly, thank you very much.

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ABSTRACT

Owing to its nutritional values, consumption of cultured marine bivalve such as P.

viridis has gained popularity in many countries including Malaysia. Besides, the use

of P. viridis as biomonitoring agent for heavy metal pollutions in coastal waters has

also been suggested. The previous study on levels of heavy metal contaminants in P.

viridis collected from Kampung Pasir Putih, Pasir Gudang, Johor was reported in

2013. Because temporal changes might have occurred at this important harvesting

site, this present research acquires public health consideration. Concentrations of

lead (Pb), cadmium (Cd) and copper (Cu) as well as mercury (Hg) in the soft tissue

of P. viridis and surface seawater sampled from the site during January to March

2015 were investigated using Flame Atomic Absorption Spectrometry (FAAS) and

Flow Injection Mercury System (FIMS), respectively. Results revealed significantly

higher concentrations of these heavy metals in P. viridis (p < 0.05) than that of

surrounding surface seawater samples. Alarmingly, the ranges of concentrations for

Pb (4.27-6.55 µg/g) and Cd (1.55-2.21 µg/g) in P. viridis mussel (wet weight)

exceeded the maximum permitted proportion prescribed by the Malaysian Food Act.

Furthermore, the concentrations of Pb (2.62-3.62 mg/L), Cd (0.72-0.78 mg/L), Cu

(0.27-0.38 mg/L) and Hg (0.21-1.49 µg/L) in surface seawater samples exceeded the

Malaysia Marine Water Quality Criteria and Standards (MMWQCS). While no

significant correlation (p > 0.05) was found between Hg (r = -0.110) in P. viridis

mussel and the surrounding surface seawater, significant correlations (p < 0.05) were

observed for Pb (r = 0.787), Cd (r = -0.620) and Cu (r = -0.794). Considering the

high concentrations of heavy metals found in both P. viridis and surface seawater at

the study site, the negative impacts on human health following consumption of this

seafood product could not be ruled out. Therefore, continuous assessment on this

aspect proves to be relevant.

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ABSTRAK

Pengambilan hasil ternakan dwicengkerang laut seperti P. viridis telah meraih

populariti di banyak negara termasuk Malaysia disebabkan oleh nilai pemakanannya.

Selain itu, penggunaan P. viridis sebagai agen biomonitor bagi pencemaran logam

berat di perairan pantai juga telah disyorkan. Kajian terdahulu ke atas bahan cemar

logam berat dalam P. viridis yang telah diambil dari Kampung Pasir Putih, Pasir

Gudang, Johor telah dilaporkan pada tahun 2013. Oleh sebab perubahan temporal

yang mungkin berlaku di tapak penternakan yang penting ini, kajian ini adalah

penting terhadap kesihatan awam. Kepekatan plumbum (Pb), kadmium (Cd) dan

kuprum (Cu) dan juga merkuri (Hg) dalam tisu lembut P. viridis dan air laut

permukaan yang disampel dari tapak tersebut dari Januari hingga March 2015 telah

dianalisis menggunakan Spektrometri Penyerapan Atom Nyala Api (FAAS) dan

Sistem Merkuri Suntikan Aliran (FIMS). Hasil kajian mendapati logam-logam berat

ini mempunyai kepekatan tinggi yang signifikan dalam P. viridis (p < 0.05)

berbanding sampel air laut permukaan. Julat kepekatan Pb (4.27-6.55 μg/g) dan Cd

(1.55-2.21 μg/g) dalam kupang P. viridis (berat basah) melebihi kadar maksimum

yang dibenarkan oleh Akta Makanan Malaysia. Tambahan pula, kepekatan Pb (2.62-

3.62 mg/L), Cd (0.72-0.78 mg/L), Cu (0.27-0.38 mg/L) dan Hg (0.21-1.49 µg/L)

dalam sampel air laut permukaan melebihi Standard dan Kriteria Kualiti Air Marin

Malaysia (MMWQCS). Tiada korelasi yang signifikan (p > 0.05) didapati antara Hg

(r = -0.110) dalam kupang P. viridis dan air laut permukaan di sekitarnya, manakala

korelasi yang signifikan (p < 0.05) dapat dilihat bagi Pb (r = 0.787), Cd (r = -0.620)

dan Cu (r = -0.794). Mengambilkira kepekatan tinggi logam berat yang terdapat

dalam kedua-dua P. viridis dan air laut permukaan di kawasan kajian, kesan negatif

ke atas kesihatan manusia berikutan pengambilan produk makanan laut ini tidak

harus dipandang remeh. Oleh itu , penilaian yang berterusan ke atas aspek ini adalah

wajar.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES x

LIST OF FIGURES xii

LIST OF SYMBOLS AND ABBREVIATIONS xiii

LIST OF APPENDICES xv

1 INTRODUCTION 1

1.1 Background of Study 1

1.2 Problem Statement 3

1.3 Objectives and Hypotheses 3

1.4 Scope of Study 5

1.5 Significance of Study 6

2 LITERATURE REVIEW 7

2.1 Perna viridis 7

2.1.1 Species Description 8

2.1.2 P. viridis as Biomonitor 10

2.1.3 Heavy Metal Contamination on P. viridis

Mussel

11

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2.2 Sources of Heavy Metals and Their Applications 12

2.3 Prevailing Knowledge on Heavy Metals Toxicities 15

2.3.1 Lead (Pb) 15

2.3.2 Cadmium (Cd) 15

2.3.3 Copper (Cu) 16

2.3.4 Mercury (Hg) 16

2.4 Maximum Levels for Heavy Metals in Molluscs 17

2.5 Water Quality Standard 18

2.5.1 Marine Water Quality Index (MWQI) 18

2.5.2 Malaysia Marine Water Quality Criteria

and Standards (MMWQCS)

19

2.6 Analysis of Heavy Metals by FAAS and FIMS 21

2.6.1 Flame Atomic Absorption Spectrometry

(FAAS)

21

2.6.2 Flow Injection Mercury System (FIMS) 22

3 MATERIALS AND METHODS 23

3.1 Chemicals and Reagents 23

3.2 Laboratory Wares 23

3.3 Instrumentation 24

3.4 Experimental Design 24

3.4.1 Description of the Sampling Area 28

3.4.2 Sample Preparation 28

3.4.3 Sample Digestion 28

3.4.3.1 Digestion of P. viridis 28

3.4.3.2 Digestion of Seawater 29

3.4.4 Analysis of Metals 29

3.4.4.1 Preparation of Standard Solutions

for FAAS and FIMS

31

3.4.4.2 Preparation of Chemical

Solutions for FIMS Analysis of

Mercury

31

3.5 Method Validation 32

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3.5.1 Linearity 32

3.5.2 Limit of Detection and Limit of

Quantitation

32

3.5.3 Percentage Recovery 33

3.6 Statistical Analysis 33

4 RESULTS AND DISCUSSION 35

4.1 Validation of Analytical Method Used for

Analysing Selected Heavy Metals in P. viridis

Mussel and Surface Water Samples

35

4.1.1 Calibration Curves, Limit of Detection and

Limit of Quantitation

35

4.1.2 Percentage Recovery of Heavy Metals 38

4.2 Heavy Metals in P. viridis Mussel and Surface

Seawater at Kampung Pasir Putih, Pasir Gudang,

Johor

38

4.2.1 Concentrations of Pb, Cd, Cu and Hg in P.

viridis Mussel and Surface Seawater

39

4.2.2 Association between the Concentrations of

Pb, Cd, Cu and Hg in P. viridis Mussel

with that of Surface Seawater

42

5 CONCLUSION AND RECOMMENDATIONS 48

5.1 Conclusion 48

5.2 Recommendations 49

REFERENCES 50

Appendix A 62

ix

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Summary of heavy metal concentrations (µg/g dw) in P.

viridis soft tissues reported from previous studies

13

2.2 Maximum levels of heavy metals (µg/g wet weight) in

molluscs set by different countries and organisations

17

2.3 Marine Water Quality Index 18

2.4 Malaysia Marine Water Quality Criteria and Standards 19

3.1 GPS coordinates of all the sampling sites at Kampung

Pasir Putih, Pasir Gudang, Johor

25

3.2 Analytical conditions for FAAS and FIMS 30

4.1 Calibration parameters, LOD and LOQ for heavy metals

analysed

37

4.2 Percentage recovery (%) of Pb, Cd, Cu and Hg in P.

viridis mussel and surface seawater samples

40

4.3 Concentration of lead (Pb) in P. viridis mussel and

surface seawater samples collected from Kampung Pasir

Putih, Pasir Gudang, Johor during January-March 2015

43

4.4 Concentration of cadmium (Cd) in P. viridis mussel and



44

4.5 Concentration of copper (Cu) in P. viridis mussel and



45

4.6 Concentration of mercury (Hg) in P. viridis mussel and



46

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4.7 Association between the concentrations of Pb, Cd, Cu

and Hg in P. viridis mussel versus that of surface

seawater in Kampung Pasir Putih, Pasir Gudang, Johor

during January-March 2015

47

xi

LIST OF FIGURES

FIGURE NO. TITLE PAGE

1.1 Conceptual framework of the research 4

2.1 The taxonomy of green mussel, P. viridis (NIMPIS,

2016)

8

2.2 P. viridis mussels: (a) Female (b) Male 9

3.1 The overall view of sampling sites and its surrounding

areas (Google Earth, 2016)

25

3.2 A map indicating the sampling sites of P. viridis

mussels at Kampung Pasir Putih, Pasir Gudang, Johor

(S1-S5) (Google Maps, 2015)

26

3.3 Close proximity of a harvesting site with the bustling

Pasir Gudang Seaport

26

3.4 Overall flowchart of the research work 27

4.1 Standard calibration curve for Pb 36

4.2 Standard calibration curve for Cd and Cu 36

4.3 Standard calibration curve for Hg 37

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LIST OF SYMBOLS AND ABBREVIATIONS

°C ‒ Degree Celsius

% ‒ Percent

Cd ‒ Cadmium

cm ‒ Centimetre

Cu ‒ Copper

DOE ‒ Department of Environment

DOF ‒ Department of Fisheries

FAAS ‒ Flame Atomic Absorption Spectrometry

Fe ‒ Iron

FIMS ‒ Flow Injection Mercury System

g ‒ Gram

Hg ‒ Mercury

LOD ‒ Limit of detection

LOQ ‒ Limit of quantification

m ‒ Metre

min ‒ Minute

µg/g ‒ Microgram per gram

µg/L ‒ Microgram per litre

µL ‒ Microlitre

mg/L ‒ Milligram per litre

mL ‒ Millilitre

mm ‒ Millimetre

MWQI ‒ Marine Water Quality Index

MMWQCS ‒ Malaysia Marine Water Quality Criteria and Standards

MΩ.cm ‒ Megohm-centimetre

ng/g ‒ Nanogram per gram

xiii

P. viridis ‒ Perna viridis

Pb ‒ Lead

ppb ‒ Parts per billion

ppm ‒ Parts per million

ppt ‒ Parts per trillion

U.S. EPA ‒ United States Environmental Protection Agency

U.S. FDA ‒ United States Food and Drug Administration

Zn ‒ Zinc

xiv

LIST OF APPENDICES

APPENDIX TITLE PAGE

A SPSS data outputs 62

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CHAPTER 1

INTRODUCTION

1.1 Background of Study

The Asian green mussel, Perna viridis is an economically important coastal

bivalve mollusc belonging to the family Mytilidae; known as the Philippine green

mussel or green-lipped mussel in certain parts of the world (National Introduced

Marine Pest Information System, NIMPIS, 2016). The mussels are commonplace

along the coastal marine waters of the Indo-Pacific region (Gosling, 2003) and at

several portions of coastal areas of Peninsular Malaysia (Ismail et al., 2000). The use

of P. viridis as a biomonitoring agent for heavy metal pollutions within the coastal

environment has been reported (Nicholson & Szefer, 2003; Hadibarata et al., 2012;

Vasanthi et al., 2012), attributable to its cost effectiveness as well as reliability (Yap

et al., 2006a). Furthermore, as a biomonitoring agent, P. viridis mussels have been

indicated as sedentary organisms, long living, easily identifiable, reasonably

abundant and available throughout the year, tolerant to environmental changes and

pollution (Wagner & Boman, 2004), as well as having good net accumulation

capacities (Yap et al., 2004a).

Considering that P. viridis mussels are made up by about 60% of protein for

every 100 g of its dry weight (Choo & Ng, 1990), having substantial amounts of

vitamins and trace elements (Gopalakrishnan & Vijayavel, 2009), while abundantly

found within the coastal region of Peninsular Malaysia; they become an important

source of nutrients for human consumption (Yap et al., 2004a). Interestingly, while

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Johor has been the largest producer of P. viridis mussels in Malaysia (Department of

Fisheries Malaysia, DOF, 2013), its major harvesting area (Kampung Pasir Putih at

the Straits of Tebrau) for this aquaculture product is located around the Pasir Gudang

Seaport and industrial areas (Yap et al., 2004b). It has been indicated that Kampung

Pasir Putih has been subjected to various industrial and socioeconomic activities,

mainly petrochemical and its related industries as well as shipping, land reclamation

and urbanization (Yap et al., 2004b). Hence, the possibility of contamination by

heavy metals from the seaport and industrial areas into the surrounding seawater

where the mussels are reared could not be ruled out. Recognising the possibilities of

contamination by heavy metals in the mussels harvested in Kampung Pasir Putih, a

number of studies conducted between 2002 to 2013, have been reported (Yap et al,

2002, 2003, 2004a, 2004b, 2004c, 2005a, 2006a, 2006b; Eugene Ng et al., 2013).

Due to the possible temporal changes in socioeconomic and environmental factors

that may have occurred at the harvesting area and since review of the literature

reveals no specific research on this aspect beyond year 2013, the current level of

safety for consuming such product could not be ascertained.

It is pertinent to indicate here that acute and/or chronic exposure towards

heavy metals (e.g. lead, Pb; cadmium, Cd; copper, Cu; mercury, Hg) would lead to

detrimental health implications among humans (Cope et al., 2004; Flanagan et al.,

2008). While acute exposure to high levels of heavy metals may result in brain

damage, paralysis, anaemia and disruption of gastrointestinal system, chronic

exposure of such contaminants has been attributable to damages of the kidneys,

reproductive, immune, nervous, respiratory and cardiovascular systems (Cope et al.,

2004; Flanagan et al., 2008). Alarmingly, even a low level of exposure may possibly

cause disturbance in the intellectual development among children. In addition,

miscarriage, stillbirth, premature birth and low birth weight, as well as

malformations of the foetus and/or infants among pregnant women those were

exposed to heavy metals have been reported (WHO, 2015). Therefore, continuous

evaluation of these contaminant levels in such aquaculture product as P. viridis

mussels, in view of its toxicological risks towards public health, merits serious

consideration.

3

1.2 Problem Statement

It is pertinent to indicate here that the last ecotoxicological assessment

covering this area of aquaculture of interest was reported in 2013 (Eugene Ng et al.,

2013). Considering the (a) role of P. viridis mussels as a cheap source of nutrients,

(b) its good net accumulation capacities of pollutants, (c) toxicities of heavy metals

towards humans and (d) temporal changes that may have occurred, continuous

assessments on the concentrations of heavy metals as contaminants, both in water

and in the mussels are paramount. Because high concentrations of Pb, Cd and Cu had

been reportedly found in mussels from the different sites in Johor (Yap et al., 2003,

2004b, 2005a, 2006b, 2007) and since awareness on toxicities of Hg exposure

associated with fish and seafood consumption has been increasing (Hajeb et al.,

2012), the choice of these four contaminants appears justifiable. Therefore, this

present research designed to assess the concentrations of Pb, Cd, Cu and Hg in the

harvested P. viridis mussels from the different sampling sites within Kampung Pasir

Putih harvesting area as well as its surrounding seawater samples at three different

intervals (January-March 2015) acquires public health consideration. The conceptual

framework of this present research is presented in Figure 1.1.

1.3 Objectives and Hypotheses

Taking into account all the relevant information discussed above, this present

research was set to achieve the following objectives:

i. Determine the concentrations of Pb, Cd, Cu and Hg in the sampled P.

viridis as well as surrounding seawater samples during January to

March 2015.

ii. Compare and correlate the concentrations of Pb, Cd, Cu and Hg in the

sampled P. viridis with that of surrounding seawater samples.

iii. Compare the differences in the concentrations of Pb, Cd, Cu and Hg

in the sampled P. viridis as well as the surrounding seawater samples

during the different sampling intervals (January-March 2015).

4

Note: Dotted lines indicate the contextual importance of the present research for

assessing contamination of Pb, Cd, Cu and Hg in P. viridis mussel and surface

seawater.

Figure 1.1: Conceptual framework of the research

Consumption of P. viridis mussel as protein source

Biomonitoring agent for marine heavy metal pollution

Assessment of heavy metal

pollution status in marine

environment

Status of contamination by Pb, Cd, Cu and

Hg in P. viridis mussel at Kampung Pasir

Putih, Pasir Gudang, Johor

Biomonitoring agent,

P. viridis

Sediment

Seawater

Assessment:

Year 2002-2013

Concentrations of Pb, Cd,

Cu and Hg in P. viridis

mussel and surface seawater

at three different intervals

(January-March 2015)

Temporal

changes

Current status?

5

In view of all the objectives listed above, it was hypothesised that:

a) The concentrations of Pb, Cd, Cu and Hg in the sampled P. viridis would

be significantly different (p < 0.05) and highly correlated with that of

surrounding seawater samples.

b) The concentrations of Pb, Cd, Cu and Hg in the sampled P. viridis and the

surrounding seawater samples would be significantly different among the

three sampling intervals (January-March 2015).

1.4 Scope of Study

This present research involved samples of P. viridis mussels as well as the

surrounding surface seawater from different sampling sites within the Kampung

Pasir Putih harvesting area of Johor (1°25'−26'N 103°55'−57'E); those were collected

at three different sampling intervals (January-March 2015). Following the analytical

methods described by previous researchers (U.S. Environmental Protection Agency,

EPA, 1992; Rahman et al., 2012; Chadid et al., 2014), samples collected were

analysed for Pb, Cd, Cu and Hg. Analysis of Pb, Cd and Cu using a Flame Atomic

Absorption Spectrometry (FAAS) (Perkin Elmer PinAAcle 900T). In addition,

analysis of Hg was done using the Flow Injection Mercury System (FIMS) (Perkin

Elmer FIMS 100). The data were presented in microgram per gram (µg/g) of sample

dry weight (dw). Partial validation (linearity, limit of detection (LOD), limit of

quantitation (LOQ) and percentage recovery) of the analytical methods used was also

attempted.

6

1.5 Significance of Study

The findings reported here would be able to elucidate the levels of

contaminations by Pb, Cd, Cu and Hg in the surrounding surface seawater as well as

P. viridis mussels harvested in one of the major harvesting areas in Johor i.e.

Kampung Pasir Putih. In this context, determining if such contaminants in the P.

viridis mussels and surrounding surface seawater samples were within the maximum

permitted proportions of metal contaminants prescribed in the Fourteenth Schedule

of the Food Act 1983 (Act 281) & Regulations (2013) for assessing its potential

threat to public health was found pertinent. The findings may be of applied values for

the environmental and health authorities for formulating suitable intervention

programmes for managing this significant issue of public interest.

50

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